Differential subcellular localization of RIC-3 Isoforms and their role in determining 5-HT3 receptor composition
ABSTRACT RIC-3 has been identified as a chaperone molecule involved in promoting the functional expression of nicotinic acetylcholine and 5-HT(3) receptors in mammalian cells. In this study, we examined the effects of RIC-3a (isoform a) and a truncated isoform (isoform d) on RIC-3 localization, mobility, and aggregation and its effect on 5-HT3 receptor composition in mammalian cells. Human RIC-3a possesses an amino-terminal signal sequence that targets it to the endoplasmic reticulum where it is distributed within the reticular network, often forming large diffuse "slicks" and bright "halo" structures. RIC-3a is highly mobile within and between these compartments. Despite the propensity for RIC-3a to aggregate, its expression enhances the level of surface 5-HT3A (homomeric) receptors. In contrast, RIC-3a exerts an inhibitory action on the surface expression of heteromeric 5-HT3A/B receptors. RIC-3d exhibits an altered subcellular distribution, being localized to the endoplasmic reticulum, large diffuse slicks, tubulo-vesicular structures, and the Golgi. Bidirectional trafficking between the endoplasmic reticulum and Golgi suggests that RIC-3d constitutively cycles between these two compartments. In support of the large coiled-coil domain of RIC-3a being responsible for protein aggregation, RIC-3d, lacking this cytoplasmic domain, does not aggregate or induce the formation of bright aggregates. Regardless of these differences, isoform d is still capable of enhancing homomeric, and inhibiting heteromeric, 5-HT3 receptor expression. Thus, both isoforms of RIC-3 play a role in determining 5-HT3 receptor composition.
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ABSTRACT: RIC-3 enhances the functional expression of certain nicotinic acetylcholine receptors (nAChRs) in vertebrates and invertebrates and increases the availability of functional receptors in cultured cells and Xenopus laevis oocytes. Maximal activity of RIC-3 may be cell-type dependent, so neither mammalian nor invertebrate proteins is optimal in amphibian oocytes. We cloned the X. laevis ric-3 cDNA and tested the frog protein in oocyte expression studies. X. laevis RIC-3 shares 52% amino acid identity with human RIC-3 and only 17% with that of Caenorhabditis elegans. We used the C. elegans nicotinic receptor, ACR-16, to compare the ability of RIC-3 from three species to enhance receptor expression. In the absence of RIC-3, the proportion of oocytes expressing detectable nAChRs was greatly reduced. Varying the ratio of acr-16 to X. laevis ric-3 cRNAs injected into oocytes had little impact on the total cell current. When X. laevis, human or C. elegans ric-3 cRNAs were co-injected with acr-16 cRNA (1 : 1 ratio), 100 μM acetylcholine induced larger currents in oocytes expressing X. laevis RIC-3 compared with its orthologues. This provides further evidence for a species-specific component of RIC-3 activity, and suggests that X. laevis RIC-3 is useful for enhancing the expression of invertebrate nAChRs in X. laevis oocytes.Journal of Neurochemistry 09/2012; 123(6). DOI:10.1111/jnc.12013 · 4.24 Impact Factor
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ABSTRACT: The α7 subtype of nicotinic acetylcholine receptors (AChRs) is one of the most abundant members of the Cys-loop family of receptors present in the central nervous system. It participates in various physiological processes and has received much attention as a potential therapeutic target for a variety of pathologies. The importance of understanding the mechanisms controlling AChR assembly and cell-surface delivery lies in the fact that these two processes are key to determining the functional pool of receptors actively engaged in synaptic transmission. Here we review recent studies showing that RIC-3, a protein originally identified in the worm Caenorhabditis elegans, modulates the expression of α7 AChRs in a subtype-specific manner. Potentiation of AChR expression by post-transcriptional events is also critically assessed.Biochimica et Biophysica Acta 03/2012; 1818(3):718-29. DOI:10.1016/j.bbamem.2011.10.012 · 4.66 Impact Factor
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ABSTRACT: RIC-3 is a transmembrane protein which enhances maturation (folding and assembly) of neuronal nicotinic acetylcholine receptors (nAChRs). In this study, we report the cloning and characterisation of 11 alternatively spliced isoforms of Drosophila melanogaster RIC-3 (DmRIC-3). Heterologous expression studies of alternatively spliced DmRIC-3 isoforms demonstrate that nAChR chaperone activity does not require a predicted coiled-coil domain which is located entirely within exon 7. In contrast, isoforms containing an additional exon (exon 2), which is located within a proline-rich N-terminal region, have a greatly reduced ability to enhance nAChR maturation. The ability of DmRIC-3 to influence nAChR maturation was examined in co-expression studies with human alpha7 nAChRs and with hybrid nAChRs containing both Drosophila and rat nAChR subunits. When expressed in a Drosophila cell line, several of the DmRIC-3 splice variants enhanced nAChR maturation to a significantly greater extent than observed with human RIC-3. In contrast, when expressed in a human cell line, human RIC-3 enhanced nAChR maturation more efficiently than DmRIC-3. The cloning and characterisation of 11 alternatively spliced DmRIC-3 isoforms has helped to identify domains influencing RIC-3 chaperone activity. In addition, studies conducted in different expression systems suggest that additional host cell factors may modulate the chaperone activity of RIC-3.Journal of Neurochemistry 07/2008; 105(5):1573-81. DOI:10.1111/j.1471-4159.2008.05235.x · 4.24 Impact Factor